FuGENE® 6 Transfection Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always bring FuGENE HD to room temperature and mix well prior to use.

Upstream tips
Always bring FuGENE HD to room temperature and mix well prior to use.

Publication protocol

NF-κB responsive luciferase assay
Cells (7.5×10 cells/well) were seeded in 24-well plates with FBS-free medium at 37°C the day before transfection. Transfection was performed with transfection reagent Fugene-6 (Roche, Basel, Switzerland) according to manufacturer's specifications. NFκB-Luc reporter construct (1 µg/well) (Stratagene, La Jolla, CA, USA) was added to cells along with plasmid CMV-β-galactosidase (CMV-β-gal, 0.2 µg/well; Promega, Madison, WI, USA) and transfection reagent (15 µl/24-well plate). CMV-β-gal served as an internal control to normalize the transfection efficiency. After 24 hr incubation, cell cultures were treated with Tan IIA (1, 3 and 10 µM) or Sal B (200 µM) for 30 min. LPS was then added to stimulate NF-κB activity for 6 hr. Cell lysates were harvested with diluted reporter lysis 5X buffer (Promega). 24-well plates were added reporter lysis buffer (100 µl/well), and 20 µl cell lysate mixed with 100 µl luciferin in white 96-well plates. Luminescence was detected by luminometer-VICTOR2 Multilabel Counter (Perkin Elmer Inc., Waltham, MA, USA), as previously reported by us , .



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Manufacturer protocol

Download the product protocol from Promega for FuGENE® 6 Transfection Reagent below.

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